Biomarkers for toxic algae

ABSTRACT

The present invention is directed toward biomarkers that identify characteristics of algae. The invention is further directed toward biomarkers that serve to identify algae species and strains of algae species as well as detect the presence of algal toxins. Additional embodiments feature methods utilizing algal biomarkers and polypeptides that can serve as biomarkers.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to biochemical methods of detecting and/orclassifying the species and/or strain of an alga and detecting thepresence of toxins of algal origin.

2. Description of the Related Art

Contamination of shellfish with toxins produced by aquatic organisms isan on-going problem with the shellfish industry and aquacultureworldwide. Bans on the sale and consumption of shellfish from discretecoastline regions are often provoked by toxic harmful algal blooms(HABs). HABs are harmful to both human consumers and the ecosystem as awhole, as toxins produced by algae can sicken and kill many forms ofaquatic organisms. Further, contamination of shellfish with algal toxinscan occur in the absence of observed HABs. Hence the need for continuoussurveillance programs to protect the public from food-borne illness dueto contaminated shellfish.

Consumption of shellfish contaminated with algal toxin can lead toparalytic shellfish poisoning (PSP), a serious and potentially fatalillness. There are several types of PSP toxins (PST): saxitoxin (STX),neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3), gonyautoxins 1 and 4(GTX1,4), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2),C-3 and C-4 (C3,4) and B-2 (GTX6) toxin [1]. STX, one of the more commonones, causes paralytic symptoms in an organism by acting as a potentsodium channel blocker [2]. PST are poisonous to organisms higher up thefood chain [3] due to the accumulation in bivalves of a range ofneurotoxins produced by several dinoflagellates, particularly those ofthe genera Alexandrium, Gymnodinium and Pyrodinium.

Rapid and reliable species identification is a requirement for bothscientific research into HABs and commercial monitoring programs for theshellfish industry. In general algae research, morphological criteriaare sufficient to classify unicellular algae to species, and to identifypotentially toxin-producing dinoflagellates. Difficulties arise,however, if morphological characteristics that distinguish one alga fromthe rest of the plankton community are lacking. For instance, somemorphospecies have proven to be consistently linked to toxicity [e.g.,A. catenella (Whedon et Kofoid) has been found to be constantly toxic],but other morphospecies such as A. tamarense (Lebour) and Balech Talyorare known to exist in both toxic and non-toxic strains [4]. Even withconsiderable time and effort, morphospecies (which exist in both toxicand non-toxic strains) might not be able to be differentiated bytraditional microscopy since they may have identical morphology. Toremedy these problems, identification methods that use molecular probesfor nucleic acids or species-specific antibodies to detect specifictoxin-producing algae strains have been developed [5,6]. However theseapproaches suffer from cross-reactivity between species and strains, aswell as the diversity of algae species, strains and their toxins.

The study and identification of PSTs in the laboratory has beenperformed using a variety of biological, biochemical and chemicalanalytical procedures. Among them, biochemical (ELISA, receptorbinding), tissue culture bioassays [7], mouse bioassays [8] orsophisticated chemical analytical alternatives (HPLC-FD [9] and LC-MS[10], etc) for routine toxin monitoring. They are configured to yieldextremely high sensitivity and specificity towards the target toxinanalyte. However, limited availability of pure toxins commercially andthe large variation in specificity of the antibody to individual toxinshampered their application.

There exists a need for new methods of identifying species and strainsof algae and detecting the presence or absence of algal toxins.

SUMMARY OF THE INVENTION

The present invention relates to biomarkers useful for determiningcharacteristics of algae and for detecting algal toxins. Thus,embodiments of the present invention include methods of using, detectingand manipulating algal biomarkers as well as the biomarkers themselves.

One aspect of the present invention relates to a method of identifyingthe toxicity of a strain of algae, comprising obtaining a profile of aplurality of proteins expressed by algae in a sample of algae of unknowntoxicity, obtaining protein profiles of non-toxic and toxic strains ofalgae, comparing said protein profile from said sample of algae ofunknown toxicity to said protein profiles of non-toxic and toxic strainsof algae and identifying said toxicity of a strain of algae based onsaid comparing of the protein profiles. Additional embodiments comprisean assay for said profile of a plurality of proteins selected from thegroup consisting of 2-DE gel electrophoresis, ELISA, HPLC, peptide massfingerprinting, matrix-assisted laser desorption ionization-time offlight mass spectrometry (MALDI-TOF MS), protein arrays and nucleic acidarrays. In some embodiments, fluorescence detection is used with saidHPLC. Additional embodiments comprise an assay for said profile of aplurality of proteins, wherein said assay uses a technology selectedfrom the group consisting of Southern blotting, nucleotide sequencing,polymerase chain reaction, mass spectroscopy and nucleic acid arrayhybridization.

Additional aspects of the invention include methods for determiningwhether an alga is a toxic strain of the algae genus Alexandrium,comprising evaluating the levels of expression of a compound selectedfrom the group consisting of a polypeptide comprising the sequenceSAEYLERLGPKDADVPFTAAPGGAEHPVTFKKRPFGILRYQPGAGMKGAMVMEIIPKSRYPGDPQGQAFSSGVQSGWVVKSINGEDVLTADFGRIMDLLDDEVADPRFSKSTALALEKQGGRLAAPVEAPLGVVFAEIPGYQGNFATLSQDGQDGFAR (SEQ ID NO: 1, hereafter calledT1) and a nucleic acid comprising a sequence encoding a polypeptidecomprising the amino acid sequence of T1 (e.g.AGTGCCGAGTACCTAGAACGACTAGGGCCCAAAGACGCGGACGTGCCCTTCACGGCCGCCCCTGGCGGCGCTGAGCACCCGGTGACCTTCAAGAAGCGGCCCTTCGGCATCTTGCGCTACCAGCCGGGCGCGGGCATGAAGGGTGCCATGGTGATGGAGATCATTCCCAAGTCGCGCTACCCCGGCGACCCCCAGGGCCAGGCGTTCTCCTCGGGCGTGCAGAGCGGATGGGTCGTCAAGTCGATCAACGGTGAGGACGTGCTGACGGCGGACTTCGGCCGCATCATGGACTTGCTGGACGACGAGGTGGCCGACCCGCGCTTCTCCAAGTCGACGGCCTTGGCCCTCGAGAAGCAGGGCGGCCGCTTGGCAGCGCCGGTGGAGGCGCCCCTCGGGGTCGTCTTCGCGGAGATCCCGGGCTACCAGGGCAACTTCGCGACGCTCAGCCAGGACGGCCAGGACGGCTTCGCGCGTTA (SEQ ID NO: 2)).

Additional embodiments of the invention include kits for carrying outthe methods of the invention. In some embodiments, kits are designed tocarry out steps in a method of identifying the toxicity of a strain ofalgae, comprising steps to analyze the proteinaceous contents of asample of algae. Additional embodiments feature kits designed to carryout steps in a method for determining whether an alga is a toxic strainof an algae species, comprising a nucleic acid selected from the groupconsisting of a nucleic acid comprising a sequence encoding apolypeptide comprising the amino acid sequence of T1 and a nucleic acidcomprising a sequence that is complementary to a sequence encoding apolypeptide comprising the amino acid sequence of T1.

In some embodiments of these methods, said alga is a strain selectedfrom the group consisting of AMKS2, AMKS3, AMKS4, AMTK4, AMTK7, AMTK3,AMTK5 and AMTK6. Some embodiments comprise an assay for said polypeptidecomprising the sequence T1, wherein the assay is selected from the groupconsisting of 2-DE gel electrophoresis, ELISA, HPLC, peptide massfingerprinting, MALDI-TOF mass spectrometry, 3′ rapid amplification ofcDNA ends (3′ RACE) cloning, protein arrays and nucleic acid arrays.Fluorescence detection is used with said HPLC in some embodiments.Additional embodiments comprise an assay for said nucleic acidcomprising a sequence encoding a polypeptide comprising the sequence T1,wherein said assay uses a technology selected from the group consistingof Southern blotting, nucleotide sequencing, 3′ RACE cloning, polymerasechain reaction, MALDI-TOF mass spectrometry and nucleic acid arrayhybridization.

Embodiments of the invention also include an isolated polypeptidecomprising the sequence T1. In some embodiments, said polypeptide is ofalgal origin. The presence of said polypeptide is indicative of acharacteristic of the alga of origin in additional embodiments. Stillmore embodiments feature said characteristic as being selected from thegroup consisting of the species to which the alga belongs, the strain towhich the alga belongs and the presence of toxin.

Additional embodiments include an isolated nucleic acid comprising asequence encoding a polypeptide comprising the sequence T1. Inadditional embodiments, said nucleic acid is obtained from algae of thegenus Alexandrium.

Further embodiments relate to an isolated nucleic acid comprising thefull-length coding sequence of the T1 protein obtained through a processcomprising designing complimentary degenerate oligonucleotide primersbased on a sequence encoding a polypeptide comprising the sequence T1,performing reverse transcription PCR on an algae RNA sample by reversetranscription PCR using said complimentary degenerate primers andisolating the full-length coding sequence of the T1 protein from otherRT-PCR products. Some embodiments feature said isolated nucleic acidwherein said isolating of said full-length coding sequence comprisesscreening genetic expression libraries comprising genetic sequence fromalgae.

Additional embodiments include a method of screening for and identifyinga compound that binds to a polypeptide comprising the sequence T1,comprising: contacting said polypeptide with said compound anddetermining whether said compound binds to said polypeptide. Someembodiments comprise said method wherein said compound is a polypeptide.Other embodiments comprise said method wherein said compound is not apolypeptide.

In further embodiments of the invention, a nucleotide array is featured,comprising a nucleotide sequence comprising a sequence encoding apolypeptide comprising the sequence T1. Additional embodiments include aprotein array comprising a polypeptide comprising the sequence T1.

One aspect of the present invention comprises a method of determining acharacteristic of an alga, comprising obtaining a sample of biologicalmaterial comprising biological material from said alga, and performingan non-morphological assay to determine the presence or absence of abiomarker for said characteristic of said alga in said sample ofbiological material. In an additional embodiment of the invention, themethod determines a characteristic selected from the group consisting ofthe species to which the alga belongs, the strain to which the algabelongs and the presence of toxin. In some embodiments of the invention,the species of algae for which characteristics are being determined isAlexandrium minutum. Additional embodiments include methods where thealgae belong to a strain of A. minutum selected from the groupconsisting of AMKS2, AMKS3, AMKS4, AMTK4, AMTK7, AMTK3, AMTK5 and AMTK6.In some embodiments, the toxin whose presence is being determined is acyclic perhydropurine saxitoxin. In further embodiments, the cyclicperhydropurine saxitoxin is selected from the group consisting ofgonyautoxin 1, gonyautoxin 2, gonyautoxin 3 and gonyautoxin 4.

Additional embodiments of the invention are methods of determining acharacteristic of an alga, comprising obtaining a sample of biologicalmaterial comprising material from said alga, and performing an assay todetermine the presence or absence of a biomarker for said characteristicof said alga in said sample of biological material, wherein saidbiological material comprises material selected from the groupconsisting of whole algae, an extract or lysate of algae and a sample oftissue from an aquatic organism. Further embodiments feature methodswherein said aquatic organism is selected from the group consisting of abivalve, an invertebrate, a fish and a mammal. Additional embodimentsinclude methods where said material from an alga is selected from thegroup consisting of proteinaceous material from algae and nucleic acidfrom algae. Some embodiments feature methods wherein said assaydetermines the presence of a molecule selected from the group consistingof an algal protein and an algal nucleic acid. In particular embodimentsof the invention, said assay for determining the presence of an algalprotein uses a technology selected from the group consisting of 2-DE gelelectrophoresis, ELISA, HPLC, peptide mass fingerprinting, 3′ RACEcloning, a protein array, a nucleic acid array and MALDI-TOF massspectrometry. Further embodiments include methods wherein fluorescencedetection is used with said HPLC. Some embodiments comprise methodswherein said assay for determining the presence of an algal nucleic aciduses a technology selected from the group consisting of Southernblotting, nucleotide sequencing, polymerase chain reaction, 3′ RACEcloning, MALDI-TOF mass spectrometry and array hybridization.

Additional embodiments comprise methods wherein said biomarker comprisesa molecule selected from the group consisting of a peptide comprisingthe sequence of a non-saxitoxin algal protein and a nucleotidecomprising genetic sequence from a gene encoding a non-saxitoxin algalprotein. Said non-saxitoxin algal protein comprises the peptide sequenceSAEYL ERLGP KDADV PFTAA AGGGE EPVVF DDRP (SEQ ID NO: 3) in some methodsof further embodiments. In some methods of further embodiments, thenon-saxitoxin algal protein comprises the sequence SAEYL ERLGP KDADVPFTAA PGGPE HPVTF DKRP (SEQ ID NO: 4). In other embodiments thenon-saxitoxin algal protein comprises the sequence SAEYL ERLGP KDADVPFTAA PGGPE HSVTF FKRP (SEQ ID NO: 5).

Additional embodiments of the invention comprise a polypeptidecomprising the N-terminal amino acid sequence ofSAEYLERLGPKDADVPFTAAPGGAEHPVTFKKRPFGILRYQPGAGMKGAMVMEIIPKSRYPGDPQGQAFSSGVQSGWVVKSINGEDVLTADFGRIMDLLDDEVADPRFSKSTALALEKQGGRLAAPVEAPLGVVFAEIPGYQGNFATLSQDGQDGFAR (SEQ ID NO: 1). Someembodiments feature said N-terminal amino acid sequence wherein saidpolypeptide is of algal origin. Further embodiments include polypeptideswherein the presence of said polypeptide is indicative of acharacteristic of the alga of origin. Additional embodiments comprisepolypeptides wherein said characteristic indicative of the alga oforigin is selected from the group consisting of the species to which thealga belongs, the strain to which the alga belongs and the presence oftoxin.

One embodiment of the present invention is an isolated polypeptidecomprising SEQ ID NO: 1. In some aspects of this embodiment, thepolypeptide is of algal origin. In other aspects of this embodiment, thepresence of said polypeptide is indicative of a characteristic of thealga of origin. For example, in some embodiments, the characteristic maybe selected from the group consisting of the species to which the algabelongs, the strain to which the alga belongs and the presence of toxin.

Another embodiment of the present invention is an isolated polypeptideselected from the group consisting of a polypeptide comprising at least10 consecutive amino acids of SEQ ID NO: 1, a polypeptide comprising atleast 20 consecutive amino acids of SEQ ID NO: 1, a polypeptidecomprising at least 30 consecutive amino acids of SEQ ID NO: 1, and apolypeptide comprising more than 30 consecutive amino acids of SEQ IDNO: 1. In some aspects of this embodiment, the polypeptide comprises asequence selected from the group consisting of AP, PG, GA, AE, EH, HP,PV, VT, TF, FK, KK and KR.

Another embodiment of the present invention is an isolated nucleic acidencoding the polypeptide of SEQ ID NO: 1. In some aspects of thisembodiment, the nucleic acid comprises SEQ ID NO: 2.

Another embodiment of the present invention is an isolated nucleic acidselected from the group consisting of a nucleic acid comprising at least10 consecutive nucleotides of SEQ ID NO: 2, a nucleic acid comprising atleast 20 consecutive nucleotides of SEQ ID NO: 2, a nucleic acidcomprising at least 30 consecutive nucleotides of SEQ ID NO: 2, anucleic acid comprising at least 40 consecutive nucleotides of SEQ IDNO: 2, a nucleic acid comprising at least 50 consecutive nucleotides ofSEQ ID NO: 2, and a nucleic acid comprising more than 50 consecutivenucleotides of SEQ ID NO: 2.

Another embodiment of the present invention is a method for determiningwhether a strain of algae is toxic comprising determining whether asample obtained from said strain of algae comprises a polypeptide of SEQID NO: 1 or a portion thereof or a nucleic acid encoding a polypeptideof SEQ ID NO: 1 or a portion thereof. In some aspects of the method, theportion of SEQ ID NO: 1 comprises a portion of SEQ ID NO: 1 selectedyfrom the group consisting of:

-   (a) a portion of SEQ ID NO: 1 which includes the sequence AP;-   (b) a portion of SEQ ID NO: 1 which includes the sequence PG;-   (c) a portion of SEQ ID NO: 1 which includes the sequence GA;-   (d) a portion of SEQ ID NO: 1 which includes the sequence AE;-   (e) a portion of SEQ ID NO: 1 which includes the sequence EH;-   (f) a portion of SEQ ID NO: 1 which comprises the sequence HP;-   (g) a portion of SEQ ID NO: 1 which includes the sequence PV;-   (h) a portion of SEQ ID NO: 1 which includes the sequence VT;-   (i) a portion of SEQ ID NO: 1 which includes the sequence TF;-   (j) a portion of SEQ ID NO: 1 which includes the sequence FK;-   (k) a portion of SEQ ID NO: 1 which includes the sequence KK; and-   (l) a portion of SEQ ID NO: 1 which includes the sequence KR.

Another embodiment of the present invention is a method for determiningwhether a strain of algae is toxic comprising determining whether asample obtained from said strain of algae comprises a polypeptide of SEQID NO: 3 or a portion thereof or a nucleic acid encoding a polypeptideof SEQ ID NO: 3 or a portion thereof. In some aspects of the method, theportion of SEQ ID NO: 3 comprises a portion of SEQ ID NO: 3 selectedfrom the group consisting of:

-   (a) a portion of SEQ ID NO: 3which includes the sequence AAA;-   (b) a portion of SEQ ID NO: 3 which includes the sequence AG;-   (c) a portion of SEQ ID NO: 3 which includes the sequence GGG;-   (d) a portion of SEQ ID NO: 3 which includes the sequence GE;-   (e) a portion of SEQ ID NO: 3 which includes the sequence EE;-   (f) a portion of SEQ ID NO: 3 which includes the sequence EP;-   (g) a portion of SEQ ID NO: 3 which includes the sequence VV;-   (h) a portion of SEQ ID NO: 3 which includes the sequence VF;-   (i) a portion of SEQ ID NO: 3 which includes the sequence FD; and-   (j) a portion of SEQ ID NO: 3 which includes the sequence DD.

Another embodiment of the present invention is a method for determiningwhether a strain of algae is toxic comprising determining whether asample obtained from said strain of algae comprises a polypeptide of SEQID NO: 4 or a portion thereof or a nucleic acid encoding a polypeptideof SEQ ID NO: 4 or a portion thereof. In some aspects of the method, theportion of SEQ ID NO: 4 is selected from the group consisting of:

-   (a) comprises a portion of SEQ ID NO: 4 which includes the sequence    GGP;-   (b) a portion of SEQ ID NO: 3 which includes the sequence PE;-   (c) a portion of SEQ ID NO: 3 which includes the sequence FD; and-   (d) a portion of SEQ ID NO: 3 which includes the sequence DK.

Another embodiment of the present invention is a method for determiningwhether a strain of algae is toxic comprising determining whether asample obtained from said strain of algae comprises a polypeptide of SEQID NO: 5 or a portion thereof or a portion thereof or a nucleic acidencoding a polypeptide of SEQ ID NO: 5 or a portion thereof. In someaspects of the method, the portion of SEQ ID NO: 5 comprises a portionof SEQ ID NO: 5 selected from the group consisting of:

-   (a) portion of SEQ ID NO: 5 which includes the sequence GGP;-   (b) a portion of SEQ ID NO: 5 which includes the sequence PE;-   (c) a portion of SEQ ID NO: 5 which includes the sequence HS;-   (d) a portion of SEQ ID NO: 5 which includes the sequence SV;-   (e) a portion of SEQ ID NO: 5 which includes the sequence FF.

Another embodiment of the present invention is a kit comprising reagentsfor determining whether a strain of algae comprises a polypeptideselected from the group consisting of the polypeptide of SEQ ID NO: 1,the polypeptide of SEQ ID NO: 3, the polypeptide of SEQ ID NO: 4, thepolypeptide of SEQ ID NO: 5 or a portion of any of the foregoingpolypeptides. In some aspects, the kit comprises an antibody which candistinguish the toxicity associated polypeptide comprising SEQ ID NO: 1from a polypeptide which is not associated with toxicity. In someaspects of the kit, the polypeptide which is not associated withtoxicity is a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In someaspects of the kit, the antibody is capable of determining whether saidstrain of algae comprises a polypeptide selected from the groupconsisting of:

-   (1) a portion of SEQ ID NO: 1 which includes the sequence AP;-   (2) a portion of SEQ ID NO: 1 which includes the sequence PG;-   (3) a portion of SEQ ID NO: 1 which includes the sequence GA;-   (4) a portion of SEQ ID NO: 1 which includes the sequence AE;-   (5) a portion of SEQ ID NO: 1 which includes the sequence EH;-   (6) a portion of SEQ ID NO: 1 which comprises the sequence HP;-   (7) a portion of SEQ ID NO: 1 which includes the sequence PV;-   (8) a portion of SEQ ID NO: 1 which includes the sequence VT;-   (9) a portion of SEQ ID NO: 1 which includes the sequence TF;-   (10) a portion of SEQ ID NO: 1 which includes the sequence FK;-   (11) a portion of SEQ ID NO: 1 which includes the sequence KK;-   (12) a portion of SEQ ID NO: 1 which includes the sequence KR;-   (13) a portion of SEQ ID NO: 3which includes the sequence AAA;-   (14) a portion of SEQ ID NO: 3 which includes the sequence AG;-   (15) a portion of SEQ ID NO: 3 which includes the sequence GGG;-   (16) a portion of SEQ ID NO: 3 which includes the sequence GE;-   (17) a portion of SEQ ID NO: 3 which includes the sequence EE;-   (18) a portion of SEQ ID NO: 3 which includes the sequence EP;-   (19) a portion of SEQ ID NO: 3 which includes the sequence VV;-   (20) a portion of SEQ ID NO: 3 which includes the sequence VF;-   (21) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (22) a portion of SEQ ID NO: 3 which includes the sequence DD;-   (23) a portion of SEQ ID NO: 4 which includes the sequence GGP;-   (24) a portion of SEQ ID NO: 3 which includes the sequence PE;-   (25) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (26) a portion of SEQ ID NO: 3 which includes the sequence DK;-   (27) a portion of SEQ ID NO: 5 which includes the sequence GGP;-   (28) a portion of SEQ ID NO: 5 which includes the sequence PE;-   (29) a portion of SEQ ID NO: 5 which includes the sequence HS;-   (30) a portion of SEQ ID NO: 5 which includes the sequence SV; and-   (31) a portion of SEQ ID NO: 5 which includes the sequence FF.

In some aspects of the kit, the kit comprises a nucleic acid probe orprimer which can distinguish a nucleic acid encoding the toxicityassociated polypeptide comprising SEQ ID NO: 1 from a nucleic acid whichencodes a polypeptide which is not associated with toxicity. In someaspects of the kit, the nucleic acid encoding the toxicity associatedpolypeptide of SEQ ID NO: 1 comprises SEQ ID NO: 2. In some aspects ofthe kit, the nucleic acid probe or primer can distinguish a nucleic acidencoding the toxicity associated polypeptide comprising SEQ ID NO: 1from a nucleic acid encoding a polypeptide selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. In someaspects of the kit, the nucleic acid probe or primer is capable ofdetermining whether said strain of algae comprises a nucleic acidencoding a polypeptide selected from the group consisting of:

-   (1) a portion of SEQ ID NO: 1 which includes the sequence AP;-   (2) a portion of SEQ ID NO: 1 which includes the sequence PG;-   (3) a portion of SEQ ID NO: 1 which includes the sequence GA;-   (4) a portion of SEQ ID NO: 1 which includes the sequence AE;-   (5) a portion of SEQ ID NO: 1 which includes the sequence EH;-   (6) a portion of SEQ ID NO: 1 which comprises the sequence HP;-   (7) a portion of SEQ ID NO: 1 which includes the sequence PV;-   (8) a portion of SEQ ID NO: 1 which includes the sequence VT;-   (9) a portion of SEQ ID NO: 1 which includes the sequence TF;-   (10) a portion of SEQ ID NO: 1 which includes the sequence FK;-   (11) a portion of SEQ ID NO: 1 which includes the sequence KK;-   (12) a portion of SEQ ID NO: 1 which includes the sequence KR;-   (13) a portion of SEQ ID NO: 3which includes the sequence AAA;-   (14) a portion of SEQ ID NO: 3 which includes the sequence AG;-   (15) a portion of SEQ ID NO: 3 which includes the sequence GGG;-   (16) a portion of SEQ ID NO: 3 which includes the sequence GE;-   (17) a portion of SEQ ID NO: 3 which includes the sequence EE;-   (18) a portion of SEQ ID NO: 3 which includes the sequence EP;-   (19) a portion of SEQ ID NO: 3 which includes the sequence VV;-   (20) a portion of SEQ ID NO: 3 which includes the sequence VF;-   (21) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (22) a portion of SEQ ID NO: 3 which includes the sequence DD;-   (23) a portion of SEQ ID NO: 4 which includes the sequence GGP;-   (24) a portion of SEQ ID NO: 3 which includes the sequence PE;-   (25) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (26) a portion of SEQ ID NO: 3 which includes the sequence DK;-   (27) a portion of SEQ ID NO: 5 which includes the sequence GGP;-   (28) a portion of SEQ ID NO: 5 which includes the sequence PE;-   (29) a portion of SEQ ID NO: 5 which includes the sequence HS;-   (30) a portion of SEQ ID NO: 5 which includes the sequence SV; and-   (31) a portion of SEQ ID NO: 5 which includes the sequence FF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains scanning electron micrographs that show the minimal andinsignificant variation in morphological features between toxic (panelA, strain AMKS2) and non-toxic (panel B, strain AMTK3) strains of thealgae A. minutum.

FIG. 2 shows high performance liquid chromatograms (HPLC) of paralyticshellfish poisoning (PST) toxin profiles from various samples of A.minutum analyzed using a Cosmosil 5C18-AR column, 250×4.6 mm, a mobilephase of 0.05M phosphate buffer (pH=7.0) containing 2 mM HAS, and a flowrate of 0.8 ml min-1: (A) standard mixture of 5 toxic strains (1=GTX4,2=GTX1, 3=dcGTX3, 4=B1, 5=dcGTX2, 6=GTX3 and 7=G; and individualstrains: (B) AMTK4, (C) AMTK7, (D) AMKS2, (E) AMKS3, (F) AMKS4.

FIG. 3 shows differential proteins expression patterns between toxic andnon-toxic algae strains. 2-DE protein profiles of 40 μg soluble proteinsof A. minutum extracted with 40 mM Tris base from: (A) toxic strainAMTK7; (B) toxic strain AMKS2; (C) toxic strain AMKS4; (D) nontoxicstrain AMTK3 and (E) Composite 2-DE protein profiles obtained by loading40 μg soluble proteins of AMKS2 and AMTK3 respectively. T1 & T2 andNT1–4 were found in toxic (T) and nontoxic (NT) strains respectively.Regions enclosed by circles in (D) and (E) are expanded and detailed inupper and lower portions of (F) respectively as indicated by arrows.

FIG. 4 shows differential protein expression patterns for toxic andnon-toxic A. minutum under different phases of growth. Selected 2-DEprotein profiles of 40 μg of soluble protein from toxic strain AMKS2cultures harvested at: (A) Day 1, (B) Day 3 and (C) Day 5; and nontoxicstrain AMTK3 cultures harvested at: (D) Day 1, (E) Day 3 and (F) Day 5in the exponential growth phase during a 5-day period in full K medium.Regions enclosed by circles in (A), (B) and (C) are expanded in (G.1),(G.2) and (G.3) respectively. Corresponding NT1–3 regions enclosed in(D), (E) and (F) are expanded in (H.1), (H.2) and (H.3) respectively.

FIG. 5 shows differential protein expression patterns for toxic andnon-toxic A. minutum under different environmental conditions. 2-DEprotein profiles from toxic strain AMKS-2 grown in: (A) Nitrate-limitedbalanced growth culture; (B) 72^(th) h of darkness for thelight-starved; (C) Phosphate-limited balanced growth culture and (D)Nutrient enriched balanced growth culture with antibiotics. Profilesfrom non-toxic strain AMTK-3 grown in: (E) Nitrate-limited balancedgrowth culture; (F) 72th h of darkness for the light-starved culture;(G) Phosphate-limited balanced growth culture and (H) Nutrient enrichedbalanced growth culture with antibiotics. T1 regions enclosed by circlesin (A), (B), (C) and (D) are expanded in (I.1), (I.2), (I.3) and (I.3)respectively. NT1–3 regions enclosed in (E), (F), (G) and (H) areexpanded in (J.1), (J.2) (J.3) and (J.4) respectively.

FIG. 6 displays a MALDI-TOF peptide mass map of the peptide mixtureobtained from in-gel tryptic digestion of (A) T1 obtained from toxicstrain AMKS-2; (B) NT1; (C) NT2 and (D) NT3 obtained from nontoxicstrain AMTK-3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides methods and compounds useful in theidentification and detection of algae species, strains and toxins.Paralytic shellfish poisoning (PSP) toxins (PST) are highly toxicnatural compounds produced by a particular class of phyoplankton knownas dinoflagellates and can accumulate in shellfish during toxic algalblooms. PSP poses a significant public health threat as well as economicloss to the shellfish industry. Rapid and reliable speciesidentification is a necessary element of harmful algal bloom (HAB)research and monitoring programs for the shellfish industry. Generally,morphological criteria can be used to classify structurally dissimilarunicellular algae to species. Difficulties arise, however, ifmorphological characteristics that distinguish particular algae from therest of the plankton community are lacking, or when both toxin-producingand non-toxic strains of an algal species exist.

Researches have found that the profiles of PST expression can be bothspecies and clone specific. The level of toxicity present within thesame species of dinoflagellates varied greatly between batches isolatedfrom different localities [11] or in the same area [12]. Early findings[13] suggested that toxin composition was a stable property and thatdifferences in PST analogue profiles had a genetic basis in two algalspecies, A. tamarense and A. catenella. Thus, a toxin profile“fingerprint” has been suggested as a biochemical characteristic todistinguish strains within the Alexandrium genus and could be regardedas a potential taxonomic biomarker [14]. However, Anderson et al [15]suggested that toxin composition is variable and reflects adaptations tonutritional and environmental conditions, and not a fixed genetic trait.

In addition to exploring which algae express toxins and when suchexpression occurs, researchers have also examined the purpose(s) forwhich toxins are expressed. Wang & Hsieh [16] reported that toxins wereproduced as secondary metabolites when growth conditions becomeundesirable. According to the work of Taroncher-Oldenburg et al. [17] ona toxic dinoflagellate A. fundyense, it was found that toxinbiosynthesis is coupled to the G1 phase of the cell cycle. They alsofound that observed variations in toxin content were a result ofincreasing periods of biosynthetic activity. Due to the harmful effectsof cyclic perhydropurine saxitoxin (STX) production on humans and theenvironment, much work has been done on the ecology and physiology ofSTX biosynthesis in several PST-producing causative algal agents as wellas on monitoring and predicting outbreaks of blooms in coastal waterscaused by these organisms. However, the molecular mechanism involved intoxin biosynthesis is virtually unknown. Studies using labeledprecursors with Alexandrium tamarense and Aphanizomenon flos-aquae haveindicated that PST toxins are derived from acetate, arginine, andmethionine [18]. Several speculative pathways have been suggested forthe biosynthesis of these unique tricyclic perhydropurine derivatives.Oshima [19] has found the enzymes N-sulphotransferase and N-oxidase,which are reportedly involved in part of the PST biosynthetic pathway,in several dinoflagellates. However, direct precursors and specificenzymes have not been identified as yet and the full biosyntheticpathway for STX remains unresolved [20]. Toxin content is generally highin the exponential growth phase, but decreasing as cultures reachstationary phase. Low temperature and low phosphate concentrations bothresult in increased cell toxicity, N limitation may cause a decrease intoxicity [15]. These studies describe general patterns of dinoflagellatetoxicity, but the physiological or biochemical mechanisms underlying theobserved variations remain unknown.

In some embodiments, the present invention provides methods andmaterials for the detailed study of which proteins are related to toxinbiosynthesis in various growth conditions and algal strains. Proteomicanalysis can monitor expression of multiple proteins simultaneously. Bycomparing proteome expression of different stages of a toxicdinoflagellate and relate that to toxin production, proteins related tothe toxin production can be found. Therefore, embodiments of the presentinvention represent important new tools for studying the physiologicaland toxicological ecology of toxic dinoflagellates by uncovering themolecular mechanism involved in the complex toxin biosynthetic pathway.Additional embodiments of the invention provide methods for detectingthe presence of PST and particular species and strains of algae.

Various embodiments of the invention use a variety of detection andanalysis techniques for polypeptides and nucleic acids that are known tothose with skill in the art. For example, in some embodiments, nucleicacids that contain some portion of sequence that is algal in origin areanalyzed using nucleic acid microarrays. These arrays were developed inthe early 1990s to assist with the mapping of the human genome byspeeding up the process of genome sequencing. Briefly, a microarrayconsists of up to thousands of DNA oligonucleotide probes fixed to asolid support in a sequential manner, each probe in a specific locationon the solid support. The probes are usually synthesized directly on thesubstrate support material and are used to interrogate complex RNA ormessage populations based on the principle of complementaryhybridization. A sample of nucleic acid containing a mixture of varioussequences can be labeled and allowed to hybridize with the DNA probes ofthe microarray. After removal of partially hybridized and unhybridizednucleic acids, the presence of nucleic acids with sequencescomplementary to the sequences of probe DNAs can be detected via theirlabels. By the positions of the labeling on the array, the identity ofthe hybridizing nucleic acids can be ascertained. Microarrays thusprovide a rapid and accurate means for analyzing nucleic acid samples.They can be used to detect trace amounts of nucleic acids and todistinguish between nucleic acids differing by as little as a singlebase, in thousands of samples simultaneously. Microarray technology hasbeen used in the laboratory for RNA detection, nucleic acids sequencingprojects and for analyzing transcription profiles of cells and tissues(Lichter, P et al. (2000) Semin Hematol 37:348–357; Tusher, V G et al.(2001) Proc Nat Acad Sci 98:5116–5121; Cook, S A and Rosenzweig, A.(2002) Circ Res 91:559–564; each one of which is hereby incorporated byreference in its entirety). Microarray technology is used in someembodiments of the invention to quickly screen and identify particularnucleic acids in biological samples comprising biological material fromalgae.

To create new methods of studying, classifying and identifying bothspecies and strains of algae, including algae that produce toxins, theprotein expression profiles of algae were examined. The presentinvention is based on the discovery of biomarkers which can be used toeasily distinguish between algae of different strains and species. Thesebiomarkers can be considered as “taxonomic biomarkers” to distinguishstrains within the same species or between species and/or as potential“toxin biomarkers” for which expression was correlated to differenttoxin production patterns in A. minutum. With regards to differentstrains of A. minutun, as detailed below in the Examples section, ourresults showed that variations in morphological features between cloneswere minimal and not significant. However, strain-specific proteinprofiles with respect to toxic and non-toxic strains of A. minutum wereobtained by two dimensional gel electrophoresis (2-DE) analysis. In someembodiments of the invention, features of the protein profiles representthe potential taxonomic biomarkers for strain and speciesdifferentiation and toxic biomarkers for the detection of toxin. Initialstudies revealed that the expression pattern of T1, a unique proteinfound in a toxic strain, in relation to different phases of the growthcycle and physiological conditions, was tightly correlated to toxinbiosynthesis in the examined algae. In some embodiments of theinvention, the expression pattern of T1 can be used as a biomarker tostudy the toxin biosynthetic mechanism in toxic dinoflagellate cells. Inadditional embodiments, T1 is used as a toxin biomarker whose detectionindicates the presence of toxin in a sample. Particular embodiments ofthe invention feature methods for the in depth study of toxinbiosynthesis and metabolism in A. minutum and other phytoplankton. Forexample, some embodiments of the invention feature protein arraytechnology to study and detect toxin biosynthesis in algae.

Various embodiments of the invention feature a variety of proteinanalysis and detection technologies. In some embodiments, for example,protein array technology is used to achieve high-throughput analysis ofbiological samples containing peptides. Protein arrays can be used todetect the presence of a known protein in a sample as well as discoverpeptides, proteins and compounds that interact with a known protein.Some embodiments of the invention use protein array technology to detectalgal proteins in biological samples. Additional embodiments of theinvention use protein array technology to screen and isolate biologicalsamples for compounds that interact with an algal protein. In someembodiments, the interacting compound is a peptide. In some embodiments,the algal protein is a peptide comprising an amino acid sequence fromthe T1 biomarker protein. Techniques and strategies for the use ofprotein arrays are known to those with skill in the art. (For a review,see MacBeath G. et al. (2000) Science 289:1760–3.)

As shown below in the Example section, we have successfully identifiedand isolated proteins which have the potential to serve as taxonomicbiomarkers for algal species or clone differentiation and as a toxinbio-indicator to study the PST biosynthetic pathway and detect thepresence of toxin. The protein profiles of different clones of algalspecies were very similar confirming that they are the same speciesdespite the discrepancy in their toxin profiles. Also, the differentialexpression patterns of toxic clones were unique and readily discernablefrom the non-toxic clones. Embodiments of the present invention willlead to the identification and characterization of strain- orspecies-specific proteins and will advance the development of fast andspecific immunological procedures to identify nuisance and toxic marinephytoplankton species such as development of whole-cellimmunofluoresence assay. An approach described in the followingexamples, of identifying toxin-related proteins using a combination ofN-terminal Edman sequencing, MALDI-TOF MS analysis of tryptic digestsbased on protein spots isolated from 2D-PAGE, 3′ RACE cloning as well asphysiological verification, also serves as a blueprint for similar workwith other toxic species in the future.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein, which can be used in the practice of the presentinvention. Indeed, the present invention is in no way limited to themethods and materials described. For purposes of the present invention,the following terms are defined below.

As used herein, the term “biological material” refers to a sample ofmatter in which some portion of the material is biological in nature. Insome embodiments of the invention, the material originates in thetissues of a living organism. In other embodiments, the material issynthetic but comprising carbon-based organic compounds. Additionalembodiments feature polypeptides and polynucleotides comprisingnaturally occurring and/or artificial, non-naturally occurring aminoacids and nucleotide bases.

“Material from an alga” refers to a substance or a molecule that at onetime was part of or synthesized by an organism classified as an alga.This material may have been obtained directly from an algae sample.Alternatively, the material may be present in non-organismal media, suchas a suspension or buffer. Additionally, “material from an alga” alsorefers to a substance or a molecule that at one time was part of orsynthesized by an organism classified as an alga which is now part of,internalized within or associated with another organism and that retainssome part of the sequence, structure or activity that the substance ormolecule had when part of or synthesized by the alga.

“Biomarker” refers to a biological substance which serves to provideclassification information regarding the species or strain of an algaand/or serves to indicate the presence or absence of another substancewhich is a “material from an alga” (see above). In some embodiments ofthe invention, the biomarker is a proteinaceous or nucleic acid-basedcompound synthesized by a particular species or strain of algae for atleast some portion of the life cycle of the algae under at least one setof conditions.

An “aquatic organism” is any form of unicellular or multicellular lifethat exists for a least some part of its lifespan within an aquaticenvironment. In some embodiments of the invention, “aquatic organism”refers to an organism that spends a minority of its lifespan in anaquatic environment. In some embodiments, avian life forms that contactaqueous environments and/or any organisms that are associated withaqueous environments are considered to be aquatic organisms.

“2-DE” refers to the separation of molecules in more than one dimension,e.g. the separation of a mixture of molecules by more than one differingcharacteristic of the molecules in the mixture. In some embodiments,“2-DE” refers to the separation of proteins and peptides through gelelectrophoresis, wherein the molecules are distinguished by molecularsize and by pH/isoelectric point characteristics. In other embodiments,molecules are distinguished by the technique using either molecular sizeor pH/isoelectric point plus some other characteristic. In additionalembodiments, two or more characteristics other than molecular size orpH/isoelectric point are used.

EXAMPLES

Materials and Methods

Unless stated otherwise, all chemicals were purchased from Sigma (USA).All solvents were at least of AR grade while most were of HPLC grade.

1. Cultivation of Alexandrium minutum

1.1. Experimental Cultures

Non-axenic cultures of eight clones of the dinoflagellate A. minutumincluding 5 toxic (AMKS2, AMKS3, AMKS4, AMTK4 and AMTK7) and 3 non-toxic(AMTK3, AMTK5 and AMTK6) strains were obtained from the Institute ofFisheries Science, National Taiwan University, Taipei 10617, Taiwan,ROC. The strains of AMTK and AMKS of A. minutum were isolated from theTungKang (TK) and Kaohsiung (KS) areas of Taiwan respectively. Theseunialgal isolates were batch cultured in K medium [23] at 20° C. under a12:12 hr light: dark photoperiod at a light intensity of approximately100–150 μmol photons m-2 s-1 provided by fluorescent lamps in a Convirongrowth chamber (Model S10H, Conviron Controlled Environments, Winnipeg,Canada) for fourteen days until the mid-exponential growth phase wasreached.

1.2. Investigation of Two Representative Strains of A. minutum Over aFive-Day Period, and Also Grown Under Nutritional and EnvironmentalStresses

AMKS2 and AMTK3 were selected to represent the toxic and non-toxicstrains of A. minutum respectively and were examined over a five-dayperiod as well as under light-, nitrogen- or phosphate-limited balancedgrowth to find a set of candidate proteins which may be concurrent withthe PST toxin production patterns. Axenic cultures of toxic and nontoxicstrains of A. minutum were also generated to exclude the potentialcontamination caused by bacterial proteins on the expression of thesecandidate proteins.

1.2.1. Investigation of Alexandrium minutum Over a 5-Day Period

Batch cultures of AMKS2 (toxic strain) and AMTK3 (non-toxic strain) ofA. minutum were prepared by inoculating 1 litre of exponentially growingcultures into 10 litres of K medium and were grown as described inSection 2.1.1. Cultures of A. minutum were synchronized by adark-induced block/release method [17]. Synchronization of theexperimental cultures was achieved by maintaining the cells incontinuous darkness for 72 hours. The cells were then entrained to thesame photoperiod regime of 12L/12D as light was turned on and the firstsample was collected immediately at 8 a.m. of Day 1. Theserepresentative cultures were sampled over a 5-day period at circadiantimes separated by 12 h (i.e. 8a.m. and 8p.m. everyday) since A. minutumgrew at the rate of 0.20 divisions d-1 in optimal environmental andnutritional conditions [24].

1.2.2. Investigation of Alexandrium minutum Grown Under Nutritional andEnvironmental Stresses

The cultures at different conditions were prepared as previouslyreported [24] and briefly described below:

A. The “seed population” was concentrated by centrifugation at 1,000×gfor 15 minutes at 22° C. (himac CR 22f, Hitachi High-Speed RefrigeratedCentrifuges, Japan) at the mid log of the exponential growth phase(about Day 14) and the pellets were rinsed twice with sterilizedseawater to avoid any carry-over of nitrogen, phosphorous or inhibitorsin the inoculum.

B. The light-limited cultures were prepared by inoculating the “seedpopulation” into normal K medium to achieve an initial cell density of1×106 cells L-1 and were maintained in continuous darkness for 72 hours.

C. The nitrogen-limited and phosphorous-limited cultures were preparedby inoculating the “seed population” into nitrogen-limited andphosphate-limited K medium respectively to achieve an initial celldensity of 1×106 cells L-1 and the cultures were incubated at normaldark/light photoperiods. The cell densities were constantly monitoreduntil the stationary phase was reached. The purpose of using stationaryphase cultures was to ensure that all carry-over of nitrogen andphosphorus in the inoculum had been used up and the growth was limitedeither by phosphate or nitrate.

D. Axenic culture of strains of AMKS2 and AMTK3 of A. minutum wereestablished by inoculating the cells into the culture medium which wassupplemented with antibacterial mixtures of 100 units/ml penicillin and100 μg/ml streptomycin (GIBCO BRL antibiotics, Cat. No. 15140-122, 100mL) for several generations and mass cultures for anlaysis were made bygrowing the cells in 5 L flask with 3 L of K medium supplemented withantibacterial mixtures as mentioned above.

2. Preparation of Extract for Proteomic Analysis and HPLC-FD Analysis

Approximately 1×10⁶ A. minutum cells were collected by centrifugation at5,000×g for 20 minutes at 22° C. (himac CR 22f, Hitachi High-SpeedRefrigerated Centrifuges, Japan) and the pellets were rinsed twice withsterilized seawater to avoid any carry-over culture medium. The pelletedcells were then kept in a −80° C. ultra-low freezer for subsequentanalysis. No sample was stored for more than 3 months.

2.1. Protein Extraction and Quantification

Water-soluble proteins were isolated as previously described [22].Briefly, with a Mircotip-probe sonifier (Model 250, Branson Ultrasonics,Danbury, Conn., USA), cells were lysed in 0.5 mL of 40 mM pre-chilled(4° C.) Tris buffer at pH 8.7 containing 30 units of endonuclease(benzonase isolated from S. marcescens, Sigma E8263). Cell debris andunbroken cells were removed by centrifugation at 22,220×g for 15 min at4° C. (Mikro 22R, Hettich, Germany). The supernatants were concentratedby ultrafiltration through an Amicon YM-3 membrane (Amicon, Bedford,Mass., USA) following the manufacturer's instructions. The extracts werethen applied to a Micro BioSpin 6 Column (Bio-Rad, Hercules, Calif.,USA) previously equilibrated with a Tris buffer (10 mM Tris-HCl, pH 7.4)containing 0.02% sodium azide following the manufacturer's instructions.Flow through from the column was collected.

2.2. Toxin Extracts from Cultures of Dinoflagellate Alexandrium minutum

The pelleted cells were homogenized in 0.03M glacial acetic acid with aMircotip-probe sonifier (Model 250, Branson Ultrasonics, Danbury, Conn.,USA). Samples were chilled on ice between bursts of less than 10seconds. Cell debris and unbroken cells were removed by centrifugationat 22,220×g for 15 min at 4° C. (Mikro 22R, Hettich, Germany). Thesupernatants were filtered with a molecular-sieve membrane with a 10,000Dalton cutoff (Amicon YM-10 membrane, Amicon, Bedford, Mass., USA)following the manufacturer's instructions. The analytical procedures asdescribed by Oshima [19], for quantification of analogues of STX:gonyautoxin I, II, III, IV (GTX1–4) were used. This methodology involvedthe use of a post-column high performance liquid chromatography (HPLC)derivatization coupled with fluorescence detection. The HPLC system wasfrom Waters Corporation, USA. A stainless-steel column of reversedphased packing (Inertsil C8, 3u, 150 mm×4.6 mm and Inertsil C8, 5u, 7.5mm×4.6 mm all-guard cartridge, Alltech, USA) was used. 7 mM periodicacid in 50 mM potassium phosphate buffer (pH9.0) was used as theoxidizing reagent and 0.5M acetic acid as the acidifying reagent. Toachieve the separation of closely related toxin peaks, an isocraticelution with a mobile phase of 2 mM sodium 1-heptanesulfonate in 10 mMammonium phosphate (pH 7.1) was used. GTX¼ and GTX ⅔ standards werepurchased form the National Research Council of Canada (NRC). Thedetection limits for individual toxins were determined to be: 17 ng forGTX1; 4 ng for GTX2; 4 ng for GTX3; 10 ng for GTX4. Variability wasfound to be less than 10%. A 60–72% recovery was usually found.Concentrations were not corrected for recovery rates.

3. 2-DE

40 μg or 150 μg of each sample was mixed with a rehydration bufferbefore being loaded onto IPG strips of linear pH gradient 4–7 (AmershamBiosciences, Hong Kong, China) for subsequent staining with silver orCoomassie Brilliant Blue R-250 respectively. Rehydration, isoelectricfocusing and equilibration were performed as previously described [22].Subsequently, SDS-PAGE was performed and proteins on the 2-DE gels were(1) visualized by silver staining for pattern comparisons; (2)electro-transferred onto a PVDF membrane for N-terminal sequencing and(3) staining with Coomassie Brilliant Blue R-250 for MALDI-TOF massspectrometry. Three 2-DE gels were performed for each condition. Unlessstated otherwise, the 2-DE gels shown are representative of the 3 gelsperformed. Protein spots were selected for quantitative analysis if theyhave the potential to serve as a biomarkers, either taxonomic or toxin,and were consistently visible in all samples from one condition. Thedensity of each spot was measured using an ImageScanner (AmershamBiosciences, Hong Kong, China) equipped with ImageMaster software fromAmersham Biosciences (Hong Kong, China). The abundance of each spot wascalculated as a percentage of the total density of all 626 spotsmeasured on each gel.

FIGS. 3 and 4 show the 2-DE differential protein expression profiles fortoxic and non-toxic algae. In these experiments the IEF of the firstdimension was over a pH range of 4.0 to 7.0. The second dimension was aSDS-PAGE in a 15.0% polyacrylamide gel.

4. MALDI-TOF Mass Spectrometry and N-Terminal Amino Acid Sequencing byEdman Degradation

Protein spots were selected to determine the peptide mass fingerprintingby a MALDI-TOF mass spectrometer (MS) (Autoflex, Bruker Daltonics,Germany) if they have the potential to serve as a “taxonomic biomarker”or “toxin biomarker” and were consistently visible in all samples fromone condition. 150 μg of proteins separated by 2-D PAGE were digested ingels according to the method described by Shevchenko and coworkers [25].The digests were desalted with Zip Tip (Millipore, Boston, Mass., USA)and subjected to analysis by MALDI-TOF MS. Calibration of the instrumentwas performed with internal standards, namely angiotensin, substance P,bombesin, trypsin autolysis fragment, and adrenocorticotropic hormonewith the respective monoisotopic masses at 1046.5 m/z, 1347.7 m/z,1620.8 m/z, 2211.1 m/z, 2465.1 m/z. Monoisotopic peptide masses wereassessed to the peptides examined and database searches were performedwith the “Protein Warehouse Program” provided by Bruker againstSWISS-PROT and TrEMBL databases. The search was limited to sample spotswith corresponding molecular weight and pI range with a mass toleranceof +/−0.2 Da. One missed cleavage per peptide was allowed and cysteineswere assumed to be carbamidomethylated with acrylamide adducts andmethionine in oxidized form.

Unidentified proteins were further characterized by N-terminalsequencing. Proteins separated by 2-D PAGE were electro-transferred ontoPVDF membranes. The PVDF membrane-bound proteins were visualized bystaining with 0.1% Coomassie Brilliant Blue R-250 in 50% aqueousmethanol for 2 min, and destained in 40% methoanol and 10% acetic acid.Selected protein spots were excised and subjected to amino acidsequencing by Edman degradation, using a Procise 492 cLC Model 610Aprotein/peptide sequencer (Applied Biosystems, Hong Kong, China). Aminoacid sequences obtained were searched either against the ProteinDataBank (PDB) or SWISS-PROT by BLAST. Settings for querying shortsequences for nearly exact matches of peptide were used.

Example 1 Morphology, PST Toxin Profiles and Differential ProteinExpression Patterns of A. minitum Isolates Under Optimal Conditions

Unialgal culture of eight clones of the dinoflagellate Alexandriumminutum were divided into two categories according to their toxicitynamely toxic and non-toxic strains. Using Balech [26] as the standard oftaxonomy of Alexandrium, the following characteristics, especially smallcell size, narrow sixth precingular plate and wide posterior sulcalplate, indicated all these isolates were A. minutum. Variation inmorphological features between the two categories was minimal and notsignificant (FIG. 1). The toxin components of different clones of A.minutum were assayed by HPLC-FD and found to be gonyautoxin 1–4 only(FIG. 2). These different strains of toxic A. minutum show a wide rangeof absolute toxicities: AMTK7, AMKS-2 and AMKS-3 are dominated by GTX-3and GTX-2 with a small amount of GTX-4 and GTX-1, while AMTK4 containedtrace and almost equal amounts of GTX-3 and GTX-2 in addition to the twomajor toxins GTX-4 and GTX-1. AMKS-4 contained small amount of GTX-4 andGTX-1 and medium amounts of GTX-3 and GTX-2 when compared with otherclones. On the other hand, strains AMTK-3, AMTK-5 and AMTK-6 were foundto be consistently nontoxic.

Proteome reference maps were established for the toxic as well asnon-toxic strains of A. minutum. In general, we found strongsimilarities in gel patterns of the arrayed proteins between the strainsof the same category, toxic or non-toxic. 2-D gels of different clonesof the same category grown under the same conditions weresuperimposable. Representative 2-D gels from toxic (FIGS. 3A to 3C) andnontoxic (FIG. 3D) strains of A. minutum are shown. A comparison ofthese 2-D maps illustrates that they shared a majority of proteins andthe relative position of similarly grouped and shaped protein spots ingels of algae from both categories suggests the majority of the proteinshave the same identity. However, significant differences were observedfor several abundant proteins when comparing the gels for toxic andnon-toxic algae. A unique abundant protein spot, T1 (with pI 4.9 and anapparent molecular mass of 20 kDa) and a cluster of proteins, T2 (withpI 5.5 and apparent molecular masses between 17.5 and 20 kDa), wereconsistently found in all toxic species (FIGS. 3A to 3C). On the otherhand, in the proteome maps of non-toxic strains, several abundantproteins, NT1 (with pI 4.7 and an apparent molecular mass of 20 kDa),NT2 (with pI 4.7 and an apparent molecular mass of 19 kDa), NT3 (with pI4.8 and an apparent molecular mass of 19 kDa) and a pair of proteins,NT4 (with pI 5.4 and apparent molecular masses between 17.0 and 22 kDa),were detected in the non-toxic strains only (FIG. 3D). Since thelocation and intensity of T1 and NT3 were very close, a composite gel(FIG. 3E) was obtained by applying equal amounts of water-solubleproteins of toxic and non-toxic strains. Regions enclosed by circles inFIGS. 3D and 3E are expanded in the upper and lower portions of FIG. 3Frespectively. T1 and NT3 were confirmed to be two different proteinspots with minute difference in apparent molecular masses and pIs. Theseparate identities of T1 and NT3 were also confirmed with a combinationof MALDI-TOF MS, enzyme digestion and Edman sequencing for internalsequences.

Discussion

A commonly accepted paradigm in the study of saxitoxin-producingdinoflagellates is that the total concentration of all toxins (toxincontent) in one isolate can vary with growth conditions, but that therelative abundance of each toxin (toxin composition) does not change[28]. The toxin profiles in the test alga were consistent when cellswere grown in optimal environmental and nutritional conditions.Different strains of A. minutum can be distinguished by their uniquetoxin profiles (different relative abundance of each toxin) and thetoxin profiles of different strains of A. minutum in this study, inwhich GTX2 and GTX 3 are major components, are similar to that of A.minutum strains from Australia [29] and Spain [30]. However, Anderson etal [15] working on A. tamarense showed conclusively that drastic changesin the relative abundance of the different PST analogues could occur inAlexandrium isolates in nutrient-stressed batches or semi-continuouscultures. Therefore, toxin profile “fingerprints” can no longer beregarded as potential taxonomic biomarkers to distinguish strains withinthe Alexandrium genus. In the above example, proteomic analysis wascarried out on 5 toxic and 3 non-toxic strains of A. minutum in order tosearch for taxonomic biomarkers for both categories (toxic andnon-toxic) for strain differentiation. A comparison of the proteomereference maps generated for toxic and non-toxic strains revealed thatvariations in differential protein expression among toxic strains on onehand or among non-toxic strains on the other were minimal and notsignificant. However, pronounced differences in protein expression wereseen when toxic strains were compared to non-toxic strains. Our resultsshow that the toxic strains and the morphologically similar non-toxicstrains can be distinguished by examination of their differentialprotein expression patterns on 2-DE gels (FIG. 3). Although toxic andnon-toxic strains shared a majority of proteins, significant differencesbetween the two catagories were seen in several abundant proteins, i.e.NT1 to NT4 in non-toxic strains and T1 to T2 in toxic strains. In thisregard, 2DB analysis can detect the presence of strain-specific proteinsfor strain differentiation. The techniques demonstrated in Example 1 areuseful for many applications, including research into the metabolism ofharmful dinoflagellates, since these strain-specific proteins serve astaxonomic and toxic biomarkers that are not influenced by thephysiological state of the cells.

Example 2 Dynamics and Differential Protein Expression Patterns ofSaxitoxin Production of A. minutum Under Different Growth Phases andDifferent Nutritional and Environmental Stresses

In our study, the total culture toxin concentration closely followed thecell concentration, increasing throughout the entire experiment duringexponential phase between Day 1 and Day 5 in optimal growth conditions.The total toxicity on Day 1, Day 3 and Day 5 is listed in Table 1 asAMKS2-1, AMKS2-3 and AMKS2-5 respectively. In N-limiting conditions(AMKS2-N), cells contained half as much toxin as those cultured undernormal conditions (AMKS-1, AMKS-3 and AMKS-5). In contrast, toxincontent in PO43—limited (AMKS2-P) and light limited (AMKS2-L) culturesincreased significantly, compared with the nitrogen-limited culture(Table 1).

TABLE 1 Toxicity measurements of toxic strains of A. minutum ToxicitySample (pgSTXeq · cell⁻¹) AMKS2-1 1.91 AMKS2-3 2.68 AMKS2-5 2.94 AMKS2-N1.12 AMKS2-L 3.09 AMKS2-P 3.04 AMKS-AB 2.35 Toxicity of toxic strainAMKS2 cultures harvested Day 1 (AMKS2-1), Day 3 (AMKS2-3) and Day 5(AMKS2-5) in the exponential growth phase during a 5-day period in fullK medium and under light-limited (AMKS2-L), nitrate-limited (AMKS2-N),phosphate-limited (AMKS2-P) and supplemented with antibiotics mixture of100 units/ml penicillin and 100 μg/ml streptomycin (GIBCO BRLantibiotics, Cat. No. 15140-122, 100 mL) in balanced growth cultures(AMKS2-AB).

The possible effects of nutritional and environmental stresses andgrowth states on the expression of these strain-specific proteinprofiles were examined by analysis of the proteomic changes of tworepresentative strains, the toxic strain AMKS2 and the non-toxic strainAMTK3 during a 5-day period as well as under light-, phosphorous, andnitrogen-limited balance growth. Exponentially growing batch cultures ofAMKS2 (toxic strain) and AMTK3 (non-toxic strain) of A. minutum weresampled for 5 days at circadian times separated by 12 h (i.e. 8 a.m. and8 p.m. everyday) since A. minutum grew at the rate of 0.20 divisions d-1in optimal environmental and nutritional conditions and completed thewhole cell-cycle in approximately 3.5 days. The general protein patternsof the two strains were quite consistent with respect to differentgrowth phases. Therefore, three representative gels were selected foreach category and are shown in FIG. 4. For the toxic strain AMKS2, T1showed a progressive increase in abundance from Day 1 to Day 5 (FIG.4G), while T2 remained relatively constant over the entire growth cycle(FIGS. 4A to 4C). This protein increased from approximately 1.42% of thetotal quantified protein in Dayl to 2.96% and 3.95% of the totalquantified proteins in Day 3 and Day 5 respectively. For the non-toxicstrain AMTK3, NT1, NT2 and NT3 increased in abundance between Day 1 andDay 3, but showed no further change in Day 5 (FIGS. 4D to 4F and 4H),while NT4 was predominant in all phases (FIGS. 4D to 4F).

The differentially expressed proteins in the two representative strains,AMKS2 and AMTK3, varied slightly under different environmental stressesand no major pattern differences could be detected (FIG. 5). However,the expression of T1 was much more abundant in light-starved (5B andI.2) and phosphate limited (5C and I.3) balanced growth cultures thannitrate limited balanced growth cultures (5A and I.1). T1 expressionincreased significantly in light starved and in P-limited balancedgrowth cultures relative to the N-limited balanced growth culture. Theexpression level of T1 decreased slightly (20%) in the N-limitedbalanced growth culture in comparison to the control culture. Incontrast, the expression of NT1, NT2 and NT3 in the non-toxic strain ofA. minutum remained constant under various stress condition (FIGS. 5E to5G); the nutritional and environmental stresses had no apparent effecton their expressions (FIG. 5H). Expression of NT1, NT2 and NT3 appearsto be completely independent of stress.

In some toxic algal cultures, bacteria living outside or inside thealgal cells are either directly or indirectly associated with phytotoxinproduction [27]. Therefore extracts prepared from axenic and non-axeniccultures of A. minutum were compared in term of their toxicity anddifferential protein expression patterns. No significant differenceswere detected either in the toxin compositions (Table 1) or differentialprotein expression patterns among the axenic (FIGS. 5D and 5H) andnon-anxenic cultures (FIG. 4).

Discussion

In nutrient replete cultures with no environmental stresses, toxincontent peaked during the exponential growth phase. Dramatic increasesand declines in toxin production were observed in P-limited andN-limited cultures respectively. Enhancement in toxicity was alsoobserved in light-starved cultures. Our results were in agreement withprevious findings on Alexandrium spp. by Anderson et al [31].

Despite the fact that no major protein expression pattern differencescould be detected among algae in different growth phases and underdifferent stresses, the relative abundance of protein, T1, of toxicstrains of A. minutum showed important fluctuation throughout thedifferent growth phases and under different stresses. Under constantgrowth conditions, levels of T1 were also found to vary in accordancewith the growth cycle (FIG. 4A to 4C) and in direct proportion to theamount of toxin produced in the cells (Table 1). Anderson et al [32]suggested that cells produced toxins at rates approximating those neededto maintain a certain amount of toxin in the daughter cells after eachcell division. Further investigation under different nutritional andenvironmental stresses revealed that T1 was up-regulated with phosphate-and light-limitation but down-regulated under nitrogen limitation. Toxinproduction appeared to be nitrogen regulated and this is again inagreement with one popular speculation that toxins might be a nitrogenstorage product (approx. 33% of PST toxin weight is NH4+)[32] and theirsynthesis requires the availability of a source of nitrogen [33]. Duringnitrogen limitation, saxitoxin synthesis must compete for scarcenitrogen atoms with other essential N-containing compounds. Low toxinproduction rates and toxin contents under N limitation presumably resultfrom competition for that element between saxitoxin and T1.Environmental enhancement in toxicity and expression of T1 were observedunder light-starved and P-limited cultures. General synthesis of majorcellular components required for cell division, such as phospholipidsneeded to make up novel cell membranes, and completion of DNAreplication both required the presence of phosphorous. Under sub-optimallight conditions and severe P-limitation, cell division ceased andprotein synthesis was reduced. Lack of competition for intracellularfree amino acid from metabolic pathways specific to cell division andgeneral protein synthesis resulted in increased concentration ofnecessary precursors and enzymes for rapid toxin synthesis. ThereforePST synthesis is promoted by phosphorous and light stresses butdepressed by nitrogen deficiency, coinciding with the expression patternof T1. A combination of the observations of algae response patternsduring different physiological conditions and growth phases indicatesthat T1 expression is significantly related to toxin production.

The expression of NT1, NT2 and NT3 of a non-toxic strain of A. minutumincreased from Day 1 to Day 5 (FIGS. 4D to 4E) and then remainedconstant most of the time (FIG. 4E to 4F). Nutritional and environmentalstresses had no apparent effect on the expression of these proteins(FIGS. 5E to G) as their expression is observed to be completelyindependent of applied stressor conditions. They do not featureprominently in algal protein expression profiles at any time in thegrowth cycle of non-toxic algae. Despite the consistency of theirexpression, there may be environmental factors that act to limit theproduction of these proteins to only certain phases of the daily cycle.Since no difference in the amino acid sequence of NT1, NT2 and NT3 werefound, they conclude that they are most likely subunits or breakdownproducts of the same protein complex.

The protein expression of NT4 of the non-toxic strain and T2 of thetoxic strain of A. minutum remained fairly constant under all growthphases and growth conditions. From these results, we conclude that theexpression of these differentially expressed proteins is a stable andsteady metabolic activity and not a transient characteristic during thegrowth stages. They are not influenced by the physiological state orgrowth phases of the test alga under optimal conditions. Therefore, theyare useful as taxonomic biomarkers to differentiate toxic and non-toxicstrains grown in optimal environmental and nutritional conditions.Furthermore, the differentially expressed protein, T1, found in toxicstrains, is of particular interest with respect to its potential use astaxonomic biomarker to differentiate toxic strains from non-toxicstrains within the same species or as a toxin biomarker to study the PSTbiosynthetic pathway and detect the presence of toxin in biologicalsamples.

The association of bacteria with dinoflagellates has been studiedbecause of the possible role of bacteria in toxin synthesis. A number ofdinoflagellates undergo sexual reproduction, passing through variouslife-cycle stages in addition to the vegetative form. The presence ofbacteria within dinoflagellates has been well established [34], buttheir interaction with toxin biosynthesis still remains unknown. Thetoxin composition (Table 1) and differential protein expression profilesobtained from the axenic cultures of A. minutum (FIGS. 5D and 5H)revealed no significant difference with their non-axenic counterparts(FIGS. 4A and 4D). These findings rule out bacterial involvement intoxin synthesis in this test alga. Evidence from others indicated [32]that toxins may be synthesized using nitrogen that is recycled withinthe cells, rather than solely using inorganic nitrogen recently takeninto the cells. This suggests that bacteria co-existing inside A.minutum might represent a “self-sustaining” source of organic nutritionto the algae in a mutualistic fashion.

The close association of T1 protein expression in toxic strains of A.minutum presents additional avenues for study of the metabolism oftoxins in algae. The T1 protein may prove to be a pre-cursor ornecessary catalyst for the production of toxin, which would provide anapproach for inhibiting the production of toxin via down-regulation ofthe expression of the T1 protein.

Example 3 Protein Identification by MALDI-TOF Mass Spectrometry andN-Terminal Amino Acid Sequencing by Edman Degradation

Tryptic digestion of 2-DE gel spots corresponding to NT1, NT2, NT3 andT1 produced several peaks, all of which were common to all spots exceptfor three peaks. Two peaks, 1258.9 Da and 2197.39 Da, were produced bydigestion of the T1 spot and one peak at around 2223.1 Da was producedby from the NT1, NT2 and NT3 spots (FIG. 6). Amino acid sequencing foundthat these proteins have minor difference in their N-terminal amino acidsequence (Table 2) and these proteins may be isoforms of the sameprotein complex. The present invention includes methods, reagents andkits for determining whether a strain of algae is toxic in which analgal sample is assessed to determine which of these minor differencesare present in the polypeptides in the sample or which of these minordifferences are present in the polypeptides encoded by nucleic acids inthe sample. For example, the methods, reagents or kits may utilize orcontain antibodies or nucleic acid probes or primers which are capableof determining which of these minor differences are present in apolypeptide in the sample or which of these minor differences areencoded by a nucleic acid present in the sample.

TABLE 2 N-terminal sequencing of proteins NT1, NT2 and NT3 from nontoxicstrain, AMTK-3 and T1 from toxic strain. AMKS-2 of Alexandrium minutum.Matching proteins in the Protein spots N-terminal amino acid sequencesprotein database NT1 SAEYL ERLGP KDADV PFTAA (SEQ ID NO: 3) A GG G E EPV V F DD RP NT2 SAEYL ERLGP KDADV PFTAA (SEQ ID NO: 4) P GG P E HP V T FDK RP NT3 SAEYL ERLGP KDADV PFTAA (SEQ ID NO: 5) P GG P E HS V T F FK RPT1 SAEYL ERLGP KDADV PFTAA (SEQ ID NO: 6) PGGAE HPVTF KDiscussion

In the present study, four proteins which have the potential to serve astaxonomic biomarkers were further characterized by a combination ofMALDI-TOF mass spectrometry and N-terminal amino acid sequencing byEdman degradation. Results revealed that NT1, NT2 and NT3 share similarpeptide mass fingerprints (PMFs), indicating that these spots areisoforms of the same proteins or closely-related proteins (FIGS. 6B to6C). Despite the fact that mass spectrum of peptide tryptic digest of T1(FIG. 6A) illustrated a different set of peptide mass fragments whencompared to NT1, NT2 and NT3, the N-terminal sequences of these spotsindicate that they are isoforms of the same protein (Table 1). Increasesor decreases in peptide mass and alterations of pI values can be due topost-translational processing. The process of how theseelectrophoretically distinct isoforms, which are characteristic ofdifferent strains, were modified in toxic and nontoxic strains is asubject of ongoing interest.

Since there is little genomic sequence data currently available fordinoflagellates, we compared proteome analysis with methods usingN-terminal Edman sequencing to analysis using MALDI-TOF MS analysis oftryptic digests based on 2D-PAGE to differentiate toxic and nontoxicstrains of A. minutum. Protein identification by PMF was overall moresuccessful than N-terminal sequencing for these two categories, sincesmall changes in sequence can change the endoprotease and exoproteasecleavage sites [35] and a different set of peptide mass fragments willbe obtained despite the proteins all having similar or identical partialamino acid sequences. The unique peptide mass fragments found in toxicand nontoxic strains can be used to elucidate the functionallysignificant structural modifications in these proteins which might helpto gain an understanding of the biochemical pathways operating in thedinoflagellates and the biosynthetic mechanism of the secondarymetabolites.

Example 4 Isolation and Characterization of Full-Length BiomarkerGenetic Coding Sequences

Through the use of 2-DE gel electrophoresis, differential proteinexpression analysis and N-terminal peptide sequencing, partialN-terminal sequences have been obtained for NT1, NT2, NT3, NT4 and T1.

Based on the N-terminal sequences obtained, degenerate oligonucleotidesfor reverse-transcription-polymerase chain reaction (RT-PCR)amplification are designed and synthesized. Total RNA is isolated fromcultures of toxic and non-toxic A. minutum strains and reversetranscribed using oligo (dT). After first strand synthesis of DNA fromthe total RNA samples is completed, PCR with degenerate oligonucleotidesis used to generate partial cDNAs for the biomarker proteins. The cDNAfragments are treated enzymatically to create blunt-ended fragments,then they are ligated to a bacterial propagation vector such as pUC8.Individual plasmid clones are sequenced to identify those withN-terminal sequences matching the partial N-terminal sequence previouslydetermined for the biomarker proteins.

Plasmids containing putative partial cDNAs of the biomarker proteins areused with established laboratory techniques known to persons with skillin the art for isolation of the remaining coding sequence(s) for thebiomarker proteins. In one approach, total RNA from algae samples areused with commercially available kits to create genomic libraries forthe algal strains. Briefly, total RNA is reverse transcribed and clonedinto commercially available vectors which permit the insertion of clonedcDNAs into a lambda bacteriophage library. The library is plated withhost bacteria and hybridization membranes are placed onto the plates andspatially marked. The hybridization membranes are lifted from the platesand treated to expose the library DNA. Partial cDNA sequences ofbiomarker genetic sequence are used to create labeled probe cDNAs andthe labeled probes are hybridized to the library hybridizationmembranes. Exposure to film reveals the locations on the hybridizationmembers where probe cDNA have hybridized with lambda phage librarygenetic material. Phage samples from the plates are isolated andreplated at a lower density. Probe hybridization followed by replatingof isolated phage at a lower density is repeated until positive singularisolated phage samples can be extracted from the plate. The librarysequences in these phage samples are then sequenced. Analysis of thegenetic sequences contained within the phage library samples will revealwhether the entire coding sequence(s) of the biomarker proteins,extending into untranslated 3′ cDNA sequence, have been obtained. Ifnot, further rounds of lambda library screenings can be performed, usinglambda library sequences that are 3′ to the sequence of the originalprobe as probes for the additional rounds of screenings. Once lambdalibrary sequences have been isolated that overlap an entire codingsequence for a biomarker protein, the sequence can be assembled into asingle expression vector containing a single contiguous coding sequencefor the protein.

Example 4A

3′ rapid amplification of cDNA ends (3′ RACE) cloning was performedaccording to methods previously described (Frohman M A, Dush M K, MartinG R. Rapid production of full-length cDNAs from rare transcripts:amplification using a single gene-specific oligonucleotide primer. ProcNatl Acad Sci USA. 1988, 85:8998–9002; Ohara O, Dorit R L, Gilbert W.One-sided polymerase chain reaction: the amplification of cDNA. ProcNatl Acad Sci USA. 1989, 86:5673–7) to obtain partial DNA sequence ofT1. Briefly, total RNA was extracted from the dinoflagellate usingTrizol reagent. cDNA was then synthesized by using the 3′-RACE-oligo-dTprimer with the following sequence: 5′-GGC CAC GCG TCG ACT AGT ACT TTTTTT TTT TTT TTT T-3′ (SEQ ID NO: 7)

3′-RACE was then performed by PCR using the gene specific primer 1(5′-CCC AAA GAC GCG GAC GTG CC-3′) (SEQ ID NO: 8) and the universalprimer (5′-GGC CAC GCG TCG ACT AGT AC-3′) (SEQ ID NO: 9), and the cDNAas template. The PCR condition used was: initial denaturation at 95° C.for 3 minutes, followed by denaturation at 94° C. for 40 seconds,annealing at 60° C. for 40 seconds and extension at 72° C. for 40seconds, for 35 cycles. The resulting PCR product was diluted 100-foldand used as template in the second round of PCR. Gene specific primer 2(5′-GCG GAC GTG CCC TTC ACG GC-3′) (SEQ ID NO: 10) and universal primer(5′-GGC CAC GCG TCG ACT AGT AC-3′) (SEQ ID NO: 9) was used in the secondround of PCR. Conditions of the second round of PCR was initialdenatured at 95° C. for 3 minutes, followed by denaturation at 94° C.for, 40 seconds, annealing at 60° C. for 40 seconds and extension at 72°C. for 40 seconds, for 35 cycles. PCR product obtained was cloned andsequenced.

With the 3′rapid-amplification-of-cDNA-ends (3′ RACE) cloning method asdescribed above, part of the DNA sequence coding for T1 was found to be:

(SEQ ID NO: 2) AGTGCCGAGTACCTAGAACGACTAGGGCCCAAAGACGCGGACGTGCCCTTCACGGCCGCCCCTGGCGGCGCTGAGCACCCGGTGACCTTCAAGAAGCGGCCCTTCGGCATCTTGCGCTACCAGCCGGGCGCGGGCATGAAGGGTGCCATGGTGATGGAGATCATTCCCAAGTCGCGCTACCCCGGCGACCCCCAGGGCCAGGCGTTCTCCTCGGGCGTGCAGAGCGGATGGGTCGTCAAGTCGATCAACGGTGAGGACGTGCTGACGGCGGACTTCGGCCGCATCATGGACTTGCTGGACGACGAGGTGGCCGACCCGCGCTTCTCCAAGTCGACGGCCTTGGCCCTCGAGAAGCAGGGCGGCCGCTTGGCAGCGCCGGTGGAGGCGCCCCTCGGGGTCGTCTTCGCGGAGATCCCGGGCTACCAGGGCAACTTCGCGACGCTCAGCCAGGACGGCCAGGACGGCTTCGCGCGTTA.

The above example represents one possible approach to obtaining the fullcoding sequence for a gene when only a small portion of 5′ end sequencehas been obtained. Additional approaches for reaching the same goal areknown to those with skill in the art.

Example 5 Production of Monoclonal Antibodies to Taxonomic and ToxicBiomarker Proteins

The previous example outlined a process for obtaining a full-lengthcoding sequence for a biomarker protein of the invention. Havingobtained this coding sequence, monoclonal antibodies are produced usingthe sequence with skills and protocols known to those with skill in theart. Monoclonal antibodies to biomarker proteins provide a powerfulresearch and diagnostic tool for exploring the metabolism and synthesisof algal toxins and detecting the presence of algal toxins in biologicalsamples. A procedure that can be used to produce monoclonal antibodiesfrom the genetic sequence(s) isolated in the previous example isoutlined below. However, there are multiple systems and protocols toaccomplish this goal and this example is not intended to limit themethods of the invention to any one approach or system. The procedureoutlined below described the production of monoclonal antibodies fromone full-length coding sequence of an algal biomarker protein. Theprocedure may be used to synthesize monoclonal antibodies from otherfull-length coding sequences of algal biomarker proteins, should suchsequences exist, as well as from partial cDNA sequences derived fromgenetic material that codes for an algal biomarker protein.

The coding sequence for an algal biomarker protein is cloned into acommercially available bacterial expression vector that will express theprotein with a 6× histidine tag at either the C-terminal or N-terminalend of the biomarker protein. The vector is transformed into E. Coli andthe tagged protein is expressed in the bacteria; the recombinantbiomarker protein is then purified after extraction from the bacteria byvirtue of commercially available Ni²⁺ resin that binds the 6× histidinetag.

BALB/c mice are immunized with the purified, recombinant biomarkerprotein in Freund's complete adjuvant. The mice receive immunizationboosts at two week intervals by injection of recombinant biomarkerprotein in Freund's incomplete adjuvant initially and with just therecombinant protein for a second and final injection. Splenocytes arecollected two days after the final injection and fused with a myelonomacell line. Screenings for hybridomas producing anti-biomarker murine Abare performed using ELISA. To provide antigen for ELISA screenings,recombinant biomarker protein from E. Coli can be used, or alternativelya mammalian or insect cell expression system, utilizing mammalianexpression vectors and COS cells or baculovirus vectors and Th5 cells,for example, can be used to produce biomarker antigen protein. Multiplemethods of expressing large quantities of recombinant proteins are knownto those with skill in the art. Hybridoma cultures that test positivefor Ab to the algal biomarker protein are then replated at limitingdilutions and retested. Testing and replated are repeated until clonalhybridoma cultures producing monoclonal antibodies to the algalbiomarker protein are produced. Clonal hybridoma cultures are then usedto produce large quantities of monoclonal Ab to the algal biomarkerprotein via techniques known to those with skill in the art. Theseantibodies can then be used in basic algae research and in commercialscreening applications for algal strains and toxins.

In some instances it may be desirable to produce antibodies which arecapable of distinguishing between the toxicity associated T1 polypeptidecomprising SEQ ID NO: 1 and proteins from non-toxic strains such as NT1,NT2 and NT3 (SEQ ID NOS: 3–5). Such antibodies can be obtained bygenerating monoclonal antibodies against one of the T1, NT1, NT2 or NT3polypeptides and assessing the ability of these antibodies to bind tothe other polypeptides to identify antibodies specific to either the T1,NT1, NT2, or NT3 polypeptides. For example, in one embodiment monoclonalantibodies may be generated against T1. The antibodies against T1 arethen placed in contact with NT1, NT2 or NT3 and their ability to bind NT1, NT2 or NT3 is assessed. Those monoclonal antibodies which bind to T1which bind with significantly lower affinity or do not bind at all toNT1, NT2 or NT3 may be used in kits for determining whether a strain ofalgae is toxic. If a sample comprising polypeptides from the strainbeing evaluated binds a T1 specific antibody which binds withsignificantly lower affinity or which does not bind at all to NT1, NT2or NT3, then the strain is a toxic strain. Likewise, if a samplecomprising polypeptides from the strain being evaluated binds to anantibody which binds NT1, NT2 or NT3 but which binds T1 with asignificantly lower affinity or which does not bind T1 at all, then thestrain being evaluated is non-toxic.

In some embodiments the antibody specifically binds to one of thefollowing polypeptides:

-   (1) a portion of SEQ ID NO: 1 which includes the sequence AP;-   (2) a portion of SEQ ID NO: 1 which includes the sequence PG;-   (3) a portion of SEQ ID NO: 1 which includes the sequence GA;-   (4) a portion of SEQ ID NO: 1 which includes the sequence AE;-   (5) a portion of SEQ ID NO: 1 which includes the sequence EH;-   (6) a portion of SEQ ID NO: 1 which comprises the sequence HP;-   (7) a portion of SEQ ID NO: 1 which includes the sequence PV;-   (8) a portion of SEQ ID NO: 1 which includes the sequence VT;-   (9) a portion of SEQ ID NO: 1 which includes the sequence TF;-   (10) a portion of SEQ ID NO: 1 which includes the sequence FK;-   (11) a portion of SEQ ID NO: 1 which includes the sequence KK;-   (12) a portion of SEQ ID NO: 1 which includes the sequence KR;-   (13) a portion of SEQ ID NO: 3 which includes the sequence AAA;-   (14) a portion of SEQ ID NO: 3 which includes the sequence AG;-   (15) a portion of SEQ ID NO: 3 which includes the sequenceGGG;-   (16) a portion of SEQ ID NO: 3 which includes the sequence GE;-   (17) a portion of SEQ ID NO: 3 which includes the sequence EE;-   (18) a portion of SEQ ID NO: 3 which includes the sequence EP;-   (19) a portion of SEQ ID NO: 3 which includes the sequence VV;-   (20) a portion of SEQ ID NO: 3 which includes the sequence VF;-   (21) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (22) a portion of SEQ ID NO: 3 which includes the sequence DD;-   (23) a portion of SEQ ID NO: 4 which includes the sequence GGP;-   (24) a portion of SEQ ID NO: 3 which includes the sequence PE;-   (25) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (26) a portion of SEQ ID NO: 3 which includes the sequence DK;-   (27) a portion of SEQ ID NO: 5 which includes the sequence GGP;-   (28) a portion of SEQ ID NO: 5 which includes the sequence PE;-   (29) a portion of SEQ ID NO: 5 which includes the sequence HS;-   (30) a portion of SEQ ID NO: 5 which includes the sequence SV; and-   (31) a portion of SEQ ID NO: 5 which includes the sequence FF.

Example 6 Screening and Identification of Proteins that Interact with aPeptide of the Invention

In some embodiments of the invention, algal proteins involved in toxinmetabolism and proteins that toxin metabolism proteins interact with canbe studied. Some embodiments use the yeast two hybrid system or avariant of this system to find and identify peptides that interact withpolypeptide sequences that comprise the peptide T1 (hereby known as “T1polypeptide”). The yeast two-hybrid system is designed to studyprotein-protein interactions in vivo (Fields and Song, 1989), and reliesupon the fusion of a bait protein to the DNA binding domain of the yeastGal4 protein. This technique is also described in the U.S. Pat. No.5,667,973 and the U.S. Pat. No. 5,283,173 (Fields et al.) the technicalteachings of both patents being herein incorporated by reference.

The general procedure of library screening by the two-hybrid assay maybe performed as described by Harper et al. (1993) or as described by Choet al. (1998) or also Fromont-Racine et al. (1997).

The bait protein or polypeptide comprises, consists essentially of, orconsists of an a polypeptide or a fragment comprising a contiguous spanof at least 4 amino acids, preferably at least 6 amino acids, morepreferably at least 8 to 10 amino acids, and more preferably at least12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide thatcomprises T1.

More precisely, the nucleotide sequence encoding the polypeptidecomprising the sequence T1 or a fragment or variant thereof is fused toa polynucleotide encoding the DNA binding domain of the GAL4 protein,the fused nucleotide sequence being inserted in a suitable expressionvector, for example pAS2 or pM3.

Then, a cDNA library is constructed in a specially designed vector, suchthat the cDNA insert is fused to a nucleotide sequence in the vectorthat encodes the transcriptional domain of the GAL4 protein. The cDNAinsert can come from a variety of sources. In some embodiments, the cDNAinsert comprises sequence from a species of algae. In some embodiments,the cDNA insert comprises sequence from a human being. Preferably, thevector used is the pACT vector. The polypeptides encoded by thenucleotide inserts of the cDNA library are termed “prey” polypeptides.

A third vector contains a detectable marker gene, such as betagalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative but nonlimiting example the two different yeast strains may be the following:

-   Y190, the phenotype of which is (MATa, Leu2–3, 112 ura3–12,    trp1–901, his3-D200, ade2–101, gal4Dgal180D URA3 GAL-LacZ, LYS    GAL-HIS3, cyh^(r));-   Y187, the phenotype of which is (MATa gal4 gal80 his3 trp1–901    ade2–101 ura3–52 leu2–3, -112 URA3 GAL-lacZmet⁻), which is the    opposite mating type of Y190.

Briefly, 20 μg of pAS2/T1-polypeptide and 20 μg of pACT-cDNA library areco-transformed into yeast strain Y190. The transformants are selectedfor growth on minimal media lacking histidine, leucine and tryptophan,but containing the histidine synthesis inhibitor 3-AT (50 mM). Positivecolonies are screened for beta galactosidase by filter lift assay. Thedouble positive colonies (His⁺, beta-gal⁺) are then grown on plateslacking histidine, leucine, but containing tryptophan and cycloheximide(10 mg/ml) to select for loss of the pAS2/T1-polypeptide plasmid butretention of pACT-cDNA library plasmids. The resulting Y190 strains aremated with Y187 strains expressing the T1 polypeptide or non-relatedcontrol proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusionsas described by Harper et al. (1993) and by Bram et al. (Bram R J etal., 1993), and screened for beta galactosidase by filter lift assay.Yeast clones that are beta gal- after mating with the control Gal4fusions are considered false positives.

In another embodiment of the two-hybrid method according to theinvention, interaction between polypeptide sequences that comprise thepeptide T1 (T1 polypeptide) or a fragment or variant thereof with algalor cellular proteins may be assessed using the Matchmaker Two HybridSystem 2 (Catalog No. K1604-1, Clontech). As described in the manualaccompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1,Clontech), the disclosure of which is incorporated herein by reference,nucleic acids encoding polypeptide sequences that comprise the peptideT1 or a portion thereof, are inserted into an expression vector suchthat they are in frame with DNA encoding the DNA binding domain of theyeast transcriptional activator GAL4. A desired cDNA, preferably algalcDNA, is inserted into a second expression vector such that they are inframe with DNA encoding the activation domain of GAL4. The twoexpression plasmids are transformed into yeast and the yeast are platedon selection medium which selects for expression of selectable markerson each of the expression vectors as well as GAL4 dependent expressionof the HIS3 gene. Transformants capable of growing on medium lackinghistidine are screened for GAL4 dependent lacZ expression. Those cellswhich are positive for both the histidine selection and the lacZ assaycontain sequences that permit, facilitate, or lead to interactionbetween a polypeptide sequence that comprises the peptide T1 and theprotein or peptide encoded by the initially selected cDNA insert;including the interacting sequences themselves. Cultures of these yeastcells are grown in quantity and the library plasmid containing theinteracting sequence is isolated. The library plasmid's cDNA insert issequenced to reveal the identity of the interacting protein (if knownand published). Alternatively, the sequence information from the cDNAinsert can be used to produce reagents for finding the rest of thesequence of the polypeptide for which the cDNA insert codes a portion ofthe amino acid sequence.

Example 7 Screening for and Identifying Compounds that Interact withPeptides of the Invention

Additional embodiments of the invention provide means to screen andidentify compounds that can bind or otherwise interact with a protein orpeptide of the invention.

The test compounds which may be used in any of the assays describedherein can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis used with peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412–421), or on beads (Lam (1991) Nature354:82–84), chips (Fodor (1993) Nature 364:555–556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865–1869) or on phage(Scott and Smith (1990) Science 249:386–390); (Devin (1990) Science249:404–406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378–6382); (Felici (1991) J. Mol. Biol. 222:301–310); (Ladnersupra.).

Determining the ability of the test compound to inhibit or increase theactivity of a polypeptide comprising the sequence T1 can beaccomplished, for example, by coupling the polypeptide or a biologicallyactive portion thereof with a radioisotope or enzymatic label such thatbinding of the polypeptide or biologically active portion thereof to itscognate target molecule can be determined by detecting the labeledpolypeptide or biologically active portion thereof in a complex. Forexample, compounds (e.g., a polypeptide comprising the sequence T1 orbiologically active portion thereof) can be labeled with 125 I, 35 S, 14C, or 3 H, either directly or indirectly, and the radioisotope detectedby direct counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. The labeled molecule is placed incontact with its cognate molecule and the extent of complex formation ismeasured. For example, the extent of complex formation may be measuredby immuno precipitating the complex or by performing gelelectrophoresis. The extent of complex formation in the presence andabsence of the test compound is compared.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a polypeptide comprising the sequence T1 orbiologically active portion thereof) to interact with its cognate targetmolecule without the labeling of any of the interactants. Interaction ofthe polypeptide comprising the sequence T1 or biologically activefragment thereof with the target molecule may be measured in thepresence or absence of the test compound to identify compounds whichincrease or decrease the extent of interaction. For example, amicrophysiometer can be used to detect the interaction of a compoundwith its cognate target molecule without the labeling of either thecompound or the target molecule. McConnell, H. M. et al. (1992) Science257:1906–1912. A microphysiometer such as a cytosensor is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize a polypeptide comprisingthe sequence T1 or its target molecule to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound toa polypeptide comprising the sequence T1, or interaction of thepolypeptide with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/T1-comprising polypeptide fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or the a polypeptide comprising the sequence T1, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads or microtitre plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of the T1-comprising polypeptide's binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either aT1-comprising polypeptide or a T1-comprising polypeptide target moleculecan be immobilized utilizing conjugation of biotin and streptavidin.Biotinylated T1-comprising polypeptides or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with aT1-comprising polypeptide or target molecules but which do not interferewith binding of the T1-comprising polypeptide to its target molecule canbe derivatized to the wells of the plate, and unbound target orT1-comprising polypeptide would be trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with aT1-comprising polypeptide or target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with aT1-comprising polypeptide or target molecule.

It would be apparent to one with skill in the art that there aremultiple other ways to use the peptides of the invention to screen forcompound that interact with the peptides of the invention. The aboveexample is not intended to limit the use of the peptides of theinvention to any of the described systems.

Example 8 Nucleic Acid Based Methods for Determining Whether a Strain ofAlgae is Toxic

In some embodiments of the present invention, a nucleic acid sample isobtained from a strain of algae to be evaluated for toxicity. Thenucleic acid sample is contacted with a nucleic acid probe or primerwhich is capable of distinguishing between nucleic acids encoding thetoxicity associated polypeptide of SEQ ID NO: 1 and nucleic acids whichdo not encode a toxicity associated polypeptide. For example, thenucleic acid which does not encode a toxicity associated polypeptide maybe a nucleic acid which encodes the NT1, NT2 or NT3 polypeptide. Thus, aprimer or probe specific to nucleic acid which encodes the T1polypeptide may be placed in contact with the sample, a hybridizationreaction or amplification reaction is performed, and the presence orabsence of hybridization or amplification is assessed. Hybridization oramplification indicates that the strain comprises a nucleic acidencoding the T1 polypeptide, thereby indicating that the strain istoxic. Likewise, a primer or probe specific to a nucleic acid encodingNT1, NT2, or NT3 may be placed in contact with the sample. Hybridizationor amplification indicates that the strain encodes NT1, NT2 or NT3,thereby indicating that the strain is not toxic.

The nucleic acid primer may be an allele specific primer which is usedin an allele specific amplification procedure. Numerous methods forconducting allele specific amplification are familiar to those skilledin the art, including the methods set forth in U.S. Pat. No. 6,638,719,U.S. Pat. No. 6,083,698 and U.S. Pat. No. 5,639,611, the disclosures ofwhich are incorporated herein by reference in their entireties. Nucleicacid primers specific for nucleic acids encoding T1 or specific fornucleic acids encoding NT1, NT2 or NT3 may be used in such allelespecific amplification procedures.

Alternatively, probes which specifically hybridize to nucleic acidsencoding T1, NT1, NT2 or NT3 may be used in a Southern blot or Northernblot procedure. Hybridization may be conducted under conditions in whicha T1 specific probe will specifically hybridize to a nucleic acidencoding T1 but will not hybridize or will hybridize to a significantlylesser degree to a nucleic acid encoding NT1, NT2 or NT3. Alternatively,hybridization may be conducted under conditions in which a NT1, NT2 orNT3 specific probe will specifically hybridize to a nucleic acidencoding NT1, NT2 or NT3 but will not hybridize or will hybridize to asignificantly lesser degree to a nucleic acid encoding T1. If a nucleicacid probe specific for T1 hybridizes to a nucleic acid sample from astrain of algae being evaluated, the strain is toxic. Alternatively, ifa nucleic acid probe specific for a nucleic acid encoding NT1, NT2 orNT3 hybridizes to the sample then the strain of algae being evaluated isnon-toxic.

In some embodiments, the primer or probe specifically amplifies orspecifically hybridizes to a nucleic acid comprising a sequence encodingone of the following polypeptides:

-   (1) a portion of SEQ ID NO: 1 which includes the sequence AP;-   (2) a portion of SEQ ID NO: 1 which includes the sequence PG;-   (3) a portion of SEQ ID NO: 1 which includes the sequence GA;-   (4) a portion of SEQ ID NO: 1 which includes the sequence AE;-   (5) a portion of SEQ ID NO: 1 which includes the sequence EH;-   (6) a portion of SEQ ID NO: 1 which comprises the sequence HP;-   (7) a portion of SEQ ID NO: 1 which includes the sequence PV;-   (8) a portion of SEQ ID NO: 1 which includes the sequence VT;-   (9) a portion of SEQ ID NO: 1 which includes the sequence TF;-   (10) a portion of SEQ ID NO: 1 which includes the sequence FK;-   (11) a portion of SEQ ID NO: 1 which includes the sequence KK;-   (12) a portion of SEQ ID NO: 1 which includes the sequence KR;-   (13) a portion of SEQ ID NO: 3which includes the sequence AAA;-   (14) a portion of SEQ ID NO: 3 which includes the sequence AG;-   (15) a portion of SEQ ID NO: 3 which includes the sequenceGGG;-   (16) a portion of SEQ ID NO: 3 which includes the sequence GE;-   (17) a portion of SEQ ID NO: 3 which includes the sequence EE;-   (18) a portion of SEQ ID NO: 3 which includes the sequence EP;-   (19) a portion of SEQ ID NO: 3 which includes the sequence VV;-   (20) a portion of SEQ ID NO: 3 which includes the sequence VF;-   (21) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (22) a portion of SEQ ID NO: 3 which includes the sequence DD;-   (23) a portion of SEQ ID NO: 4 which includes the sequence GGP;-   (24) a portion of SEQ ID NO: 3 which includes the sequence PE;-   (25) a portion of SEQ ID NO: 3 which includes the sequence FD;-   (26) a portion of SEQ ID NO: 3 which includes the sequence DK;-   (27) a portion of SEQ ID NO: 5 which includes the sequence GGP;-   (28) a portion of SEQ ID NO: 5 which includes the sequence PE;-   (29) a portion of SEQ ID NO: 5 which includes the sequence HS;-   (30) a portion of SEQ ID NO: 5 which includes the sequence SV; and-   (31) a portion of SEQ ID NO: 5 which includes the sequence FF.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

REFERENCES

Each of the following references is incorporated herein by reference inits entirety:

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1. An isolated polypeptide comprising SEQ ID NO:
 1. 2. The polypeptideof claim 1, wherein said polypeptide is of algal origin.
 3. Thepolypeptide of claim 2, wherein the presence of said polypeptide isindicative of a characteristic of the alga of origin.
 4. The polypeptideof claim 3, wherein said characteristic is selected from the groupconsisting of the species to which the alga belongs, the strain to whichthe alga belongs and the presence of toxin.
 5. A method for determiningwhether a strain of algae is toxic comprising determining whether asample obtained from said strain of algae expresses the polypeptide ofclaim 1.